CN101530419A - Application of dammarane triterpenes derivative and drug composition containing dammarane triterpenes derivative - Google Patents
Application of dammarane triterpenes derivative and drug composition containing dammarane triterpenes derivative Download PDFInfo
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Abstract
The invention provides the application of a dammarane triterpenes derivative with the structure shown as the following formula (I), a drug composition containing the dammarane triterpenes derivative, and application thereof. The dammarane triterpenes derivative and the drug composition which can induce the apoptosis of hepatic stellate cells can be used for preparing drugs for preventing or treating hepatic fibrosis and hepatocirrhosis.
Description
Technical field
The present invention relates to the pharmaceutical chemistry field, specifically, the present invention relates to the purposes of dammarane's triterpenes derivant and comprise the pharmaceutical composition of this derivant.
Background technology
Hepatic fibrosis is a chronic hepatopathy important pathological feature.The causes of disease such as virus, ethanol, autoimmune disease all can cause hepatic necrosis, regeneration and persistence fibroplasia, finally cause liver cirrhosis.Confirmed that now hepatic fibrosis is reversible pathological changes, liver cirrhosis then is irreversible.Therefore, in the therapeutic process of chronic hepatopathy, the control of hepatic fibrosis occupies critical role.
People treat and/or prevent the medicine of hepatic fibrosis always in searching in recent decades, and have developed the medicine of some treatment hepatic fibrosis, as colchicine, poly phenolic acid of Radix Salviae Miltiorrhizae, soft liver sheet, interferon and hepatocyte growth-promoting factors etc.But these medicines do not reach desirable therapeutic owing to reason such as side effect or result of use be not good.Therefore, the medicine of research and development novel anti hepatic fibrosis is very necessary.
Herb Gynostemmae Pentaphylli Gynostemma pentaphyllum (Thunb.) Makino belongs to Cucurbitaceae Gynostemma, herbaceous species all herbal medicine.Pleasantly sweet because of it, at Japan " Herb Gynostemmae Pentaphylli " by name.In bright clear herbal for Relief of Famines, be the wild plant that allays one's hunger famine year with regard to Herb Gynostemmae Pentaphylli on the books.Nagai etc. at first got panoxadiol (Panxad io l) and 2 Alpha-hydroxy panoxadiol in 1976 from this plant saponin acid hydrolysis products [the 23 for Nagai, M.et al.Abstracts of Papers
RdMeeting ofthe Japanese Society of Pharmacognosy, Hiroshima, 1976,37].After this, kind of saponin surplus Chinese scholars has isolated 100 from Herb Gynostemmae Pentaphylli.Studies have shown that according to modern pharmacology Herb Gynostemmae Pentaphylli has tangible strong effect and antifatigue effect, prolong cell survival, blood fat reducing, blood sugar level strengthens the human body immunocompetence, and can prevent and treat more than 20 kind of cancer and other multiple disease.
In recent years, research worker finds that Chinese medicine Herb Gynostemmae Pentaphylli total glycosides (being called for short GP) has effect of anti hepatic fibrosis preferably.GP can obviously suppress carbon tetrachloride and cause the rat acute liver damage, significantly suppresses SGPT, and the rising of SGOT obviously descends the MDA level, SOD level obviously raise [Zhang Yulin, Jiang Xiaoping, the Yellow River rapids, Shi Zhixin, the Henan traditional Chinese medical science, 2001,21 (6), 27-28]; Reduce hepatocyte DNA synthesis rate [Hu Baochun, Li Xiaoqing, Ceng Mingxin, the sun style of study, modern digestion disease and scope magazine, 1998,3 (1), 31-34], obviously suppress the expression [Du Yanyan of composition such as P450 in the hepatocyte microsome enzyme system, Mo Ziyao, Mai Huixia, occupational medicine, 1996,23 (3), 8-10], have liver function protecting, remove free radical, lipoid peroxidization resistant; Pathological section and microscopy observed result show, GP can make indexs such as vacuolar degeneration of hepatic cell, inflammatory infiltration, necrosis significantly take a turn for the better, and the acute liver damage tool is improved significantly, and have liver detoxification function, liver cell regeneration there are certain facilitation [old several perfume (or spice), Zhang Jianguo, Zhang Li, Liu Jingbo, drug research, 2007,16 (13), 7-8].
Though people confirm Herb Gynostemmae Pentaphylli the control hepatic fibrosis are had significant effect, but it is well-known, the characteristics of Chinese medicine be its chemical constituent (comprising active component) often with the place of production, weather, cultivating and growing conditions etc. are closely related, curative effect also changes with the variation of these conditions naturally, and the Chinese herbal medicine Herb Gynostemmae Pentaphylli also has same characteristics.For example: we have done the HPLC-UV-ESI-MS analysis respectively to the 3 kinds of Herb Gynostemmae Pentaphylli total glycosides extracts (deriving from the product that Zhejiang, Guangxi and Hunan produce) that can buy on the market, the HPLC appearance time that found that the Herb Gynostemmae Pentaphylli extract that various places produce differs greatly, and illustrates that the Herb Gynostemmae Pentaphylli chemical constituent difference of each real estate is very big.
And existing research (see before and state) all is the pharmacology activity research that carries out anti-hepatic fibrosis at the total saponins of the Herb Gynostemmae Pentaphylli in the single place of production, its limitation clearly, which kind of chemical compound what can't further specify in the Herb Gynostemmae Pentaphylli total glycosides performance effect of anti hepatic fibrosis is, can not fundamentally solve probabilistic problem of the curative effect that the difference of the place of production, planting conditions brings, be unfavorable for Herb Gynostemmae Pentaphylli further is developed as anti-hepatic fibrosis new drug than high technology content and using value.
It is generally acknowledged that hepatic stellate cell is brought into play pivotal role in the generating process of hepatic fibrosis, effectively intervene the biological behaviour of hepatic stellate cell and then can successfully prevent and treat hepatic fibrosis.The activation of hepatic stellate cell is in the center in hepatic fibrosis develops, suppressing hepatic stellate cell activator or inducing the hepatic stellate cell apoptosis is one of main direction of exploitation hepatic fibrosis control medicine.
At the limitation of existing research, the applicant is off the beaten track, has found active dammarane's triterpene saponin from China Chinese medicine Herb Gynostemmae Pentaphylli.With active dammarane's triterpene saponin be lead compound we launched structural modification and structure and active relation research, and utilize hepatic stellate cell screening model commonly used at present to screen, found that dammarane's triterpenes derivant can be used as the hepatic stellate cell antiblastic, opened up a new road for preventing and treating hepatic fibrosis and liver cirrhosis.
Summary of the invention
Purpose of the present invention is for providing the purposes of dammarane's triterpenes derivant.
A further object of the present invention is for providing the pharmaceutical composition that comprises above-mentioned dammarane's triterpenes derivant and the purposes of said composition.
For achieving the above object, the invention provides the application of dammarane's triterpenes derivant in preparation prevention or the hardened medicine of treatment hepatic fibrosis regulating liver-QI with following general formula (I) expression,
Wherein, R
1Be selected from H, OH, carbonyl (O=), C
1-C
10Acyloxy or arabinose, galactose, glucose, mannose, rhamnose, ribose, xylose, or the oligonucleotide chain formed by glycosidic bond of the arabinose of esterification, galactose, glucose, mannose, rhamnose, ribose, xylose and at least two sugar or sugar ester; R
2Be selected from C
1-C
5Alkyl or C
1-C
5Thiazolinyl.
The present invention also provides a kind of pharmaceutical composition of anti-hepatic fibrosis, and it comprises dammarane's triterpenes derivant of above-mentioned general formula (I) expression.
The present invention also provides a kind of pharmaceutical composition of anti-hepatic fibrosis, and it comprises the gynostemma pentaphyllum herb extract, and described gynostemma pentaphyllum herb extract contains dammarane's triterpenes derivant of above-mentioned general formula (I) expression.The gynostemma pentaphyllum herb extract can be freeze-dried powder form or liquid form.
The present invention also provides the application of above-mentioned two kinds of pharmaceutical compositions in preparation prevention or the hardened medicine of treatment hepatic fibrosis regulating liver-QI.
The present invention implements through the following steps:
Dammarane's triterpenes derivatives monomer provided by the invention extraction separation from product cucurbitaceous plant Herb Gynostemmae Pentaphylli Gynostemma pentaphyllum (Thunb.) the Mak. herb of Hunan obtains, but contain above-mentioned dammarane's triterpenes derivatives monomer equally in the Herb Gynostemmae Pentaphylli plant that produce in other areas, also can make by same extracting method.Its preparation process is as follows:
The Hunan is produced gynostemma pentaphyllum herb and is distinguished reflux, extract, 3 times through petroleum ether, methanol, and each 3 hours, with the methanol extract liquid concentrating under reduced pressure, through silica gel (200-300 order) column chromatography, with CHCl
3-CH
3The OH gradient elution, with the 100:20 eluting fraction through Sephadex LH-20 (CH
3The OH eluting) column chromatography, again through the MCI post with CH
3OH-H
2The O gradient elution, after the ODS column chromatography, get 3 β, 20S, 23-trihydroxy Da Maji-24-alkene-21-acid-21,23-lactone 3-O-[α-L-rhamnopyranosyl (1 → 2)] [β-D-xylopyranosyl (1 → 3)]-β-D-6-O-acetyl pyranglucoside (chemical compound 1), 3 β, 20R, 23-trihydroxy Da Maji-24-alkene-21-acid-21,23-lactone 3-O-[α-L-rhamnopyranosyl (1 → 2)] [β-D-xylopyranosyl (1 → 3)]-β-D-6-O-acetyl pyranglucoside (chemical compound 2), 3 β, 20S, 23-trihydroxy Da Maji-24-alkene-21-acid-21,23-lactone 3-O-[α-L-rhamnopyranosyl (1 → 2)] [β-D-pyranglucoside (1 → 3)]-β-D-6-O-xylopyranosyl (chemical compound 3), 3 β, 20R, 23-trihydroxy Da Maji-24-alkene-21-acid-21,23-lactone 3-O-[α-L-acetyl rhamnopyranosyl (1 → 2)] [β-D-xylopyranosyl (1 → 3)]-β-D-6-O-acetyl pyranglucoside (chemical compound 4).
Chemical compound 5 and 6 is carried out chemical conversions such as esterification, oxidation or reduction, obtain chemical compound 7-15.
The structural formula of chemical compound 1-15 is as follows:
Dammarane's triterpenes derivant of the present invention has the activity that suppresses hepatic stellate cell LX-2 propagation, and it can be used as the hepatic stellate cell antiblastic, is used for prevention and treatment hepatic fibrosis, thus the formation of prevention liver cirrhosis.In addition, the pharmaceutical composition that not only comprises purified form dammarane triterpenes derivant has the activity that suppresses hepatic stellate cell LX-2 propagation, contain the plant extract that comprises dammarane's triterpenes derivant such as the pharmaceutical composition of gynostemma pentaphyllum herb extract, also have the activity that suppresses hepatic stellate cell LX-2 propagation, therefore also can be used for preparation prevention and the hardened medicine of treatment hepatic fibrosis regulating liver-QI.
Description of drawings
Fig. 1 is haematoxylin-Yihong (HE) colored graph in the test example 2;
Fig. 2 is Masson three collagen stainings in the test example 2;
Fig. 3 is α SMA immunohistochemistry figure in the test example 2;
Fig. 4 (A) is the content sketch map of mice serum aspartate transaminase in the test example 2;
Fig. 4 (B) is the content sketch map of mice serum alanine transaminase in the test example 2;
Fig. 5 is the content sketch map of hepatic tissue hydroxyproline in the test example 2.
The specific embodiment
Embodiment 1:
Gynostemma pentaphyllum herb 1000 gram is through 5L petroleum ether reflux, extract, 3 times, and each 3 hours, the gained medicinal residues extracted 3 times through methanol eddy again, each 3 hours.The methanol extract liquid concentrating under reduced pressure is got Herb Gynostemmae Pentaphylli extract extractum 70g, Herb Gynostemmae Pentaphylli is extracted extractum and silica gel mixed sample, with chloroform: methanol 100:10-100:50 carries out gradient column chromatography eluting, with 100:20 eluting fraction concentrating under reduced pressure, 15g, again through Sephadex LH-20 with the MeOH eluting to remove flavone compound, again through the MCI resin column with water: acetone 9:1-2:1 gradient elution, merge 9:1 eluting fraction reclaim under reduced pressure and get 1g, again with MeOH:H
2O=6:4 is through RP-C
18Eluting gets chemical compound 1 pure product 320mg, yield 0.46%.Merge MCI resin 8:1 eluting fraction reclaim under reduced pressure and get 1.2g, again with MeOH:H
2O=55:45 is through RP-C
18Eluting gets chemical compound 2 pure product 260mg, yield 0.37%.Merge MCI resin 7:1 eluting fraction reclaim under reduced pressure and get 0.9g, again with MeOH:H
2O=6:4 is through RP-C
18Eluting gets chemical compound 3 pure product 130mg, yield 0.19%.Merge MCI resin 6:1 eluting fraction reclaim under reduced pressure and get 1.6g, again with MeOH:H
2O=7:3 is through RP-C
18Eluting gets chemical compound 4 pure product 10mg, yield 0.01%.
Embodiment 2
The chemical compound 1 (1g) that embodiment 1 is obtained was through 2N hydrochloric acid hydrolysis 2 hours, distribute with equal-volume ethyl acetate and water, after three extractions, organic facies is merged, after saturated aqueous sodium carbonate, saturated aqueous common salt are washed successively, spend the night with anhydrous sodium sulfate drying again.Concentrating under reduced pressure gets chemical compound 5 crude product 490mg, through silicagel column with petroleum ether: acetone=9:1 eluting, chemical compound 5 pure product 250mg, yield 48.3%.
Embodiment 3
The chemical compound 2 (1g) that embodiment 1 is obtained was through 2N hydrochloric acid hydrolysis 2 hours, distribute with equal-volume ethyl acetate and water, after three extractions, organic facies is merged, after saturated aqueous sodium carbonate, saturated aqueous common salt are washed successively, spend the night with anhydrous sodium sulfate drying again.Concentrating under reduced pressure gets chemical compound 6 crude product 451mg, through silicagel column with petroleum ether: acetone=8:1 eluting, chemical compound 6 pure product 210mg, yield 40.5%.
Embodiment 4
Chemical compound 6 (30mg) is added in the mixed liquor of 0.5mL anhydrous pyridine and 0.5mL chloroacetic chloride, after the stirring at normal temperature 2 hours, add the frozen water cessation reaction, with the equal-volume ethyl acetate extraction, organic facies is washed 3 times with sour water, after saturated sodium carbonate solution, saturated aqueous common salt are washed successively, spend the night again with anhydrous sodium sulfate drying.Concentrating under reduced pressure gets chemical compound 7 crude product 25mg, through silicagel column with petroleum ether: acetone=9:1 eluting, chemical compound 7 pure product 22mg, yield 67.1%.
In the same way by chemical compound 5 preparation chemical compounds 8, yield 67.3%.
Embodiment 5
Chemical compound 6 (30mg) is added in the mixed liquor of 0.5mL anhydrous pyridine and 0.5mL propionic andydride, stirring at normal temperature 2 hours, add the frozen water cessation reaction, with the equal-volume ethyl acetate extraction, organic facies is washed 3 times with sour water, after saturated sodium carbonate solution, saturated aqueous common salt are washed successively, spend the night again with anhydrous sodium sulfate drying.Concentrating under reduced pressure gets chemical compound 9 crude product 27mg, through silicagel column with petroleum ether: acetone=9:1 eluting, chemical compound 9 pure product 24mg, yield 71.2%.
In the same way by chemical compound 5 preparation chemical compounds 10, yield 69.8%.
Embodiment 6
Chemical compound 6 (30mg) is added in the mixed liquor of 0.5mL anhydrous pyridine and 0.5mL Benzenecarbonyl chloride., stirring at normal temperature 2 hours, add the frozen water cessation reaction, with the equal-volume ethyl acetate extraction, organic facies is washed 3 times with sour water, after saturated sodium carbonate solution, saturated aqueous common salt are washed successively, spend the night again with anhydrous sodium sulfate drying.Concentrating under reduced pressure gets chemical compound 11 crude product 25mg, with petroleum ether acetone 9:1 eluting, gets chemical compound 11 pure product 23mg, yield 62.8% through silicagel column.
In the same way by chemical compound 5 preparation chemical compounds 12, yield 64.9%.
Embodiment 7
Chemical compound 6 (30mg) is dissolved in the 30ml dichloromethane, adds pyridinium chlorochromate drone salt (PCC) 30mg, stirring at normal temperature 2 hours is washed 3 times with the equal-volume sour water, after saturated sodium carbonate solution, saturated aqueous common salt are washed successively, spends the night with anhydrous sodium sulfate drying again.Concentrating under reduced pressure gets chemical compound 13 crude product 28mg, through silicagel column with petroleum ether: acetone=9:1 eluting, chemical compound 13 pure product 27mg, yield 89.6%.
In the same way by chemical compound 5 preparation chemical compounds 14, yield 87.5%.
Embodiment 8
Chemical compound 6 (30mg) is dissolved in the 5mL methanol, adds 10mg Pd/C, place H
2In the atmosphere, stirring at normal temperature 3 hours, with kieselguhr elimination Pd/C powder, distilling under reduced pressure concentrates, chemical compound 15 crude product 27mg, through silicagel column with petroleum ether: acetone=9:1 eluting, chemical compound 15 pure product 25mg, yield 83.1%.
Preparation example with above-listed dammarane's triterpenes derivant is for referencial use, and other dammarane's tetraterpene derivatives also can be according to method for preparing.
Embodiment 9
1000 gram gynostemma pentaphyllum herbs are added water boil extract 3 times, each 2 hours, merge extractive liquid,, filter AB8 macroporous resin on the rear filtrate, treat that effluent is saturated after, water flushing pillar is colourless to effluent, use 90% ethanol elution then, the fraction concentrating under reduced pressure gets gynostemma pentaphyllum herb extract 1658.3 grams.Detect with high-efficient liquid phase technique (HPLC-ELSD), the content that records chemical compound 1 and chemical compound 2 is respectively 3.9%, 4.2%.
Embodiment 10
1000 gram gynostemma pentaphyllum herbs are added 40% alcohol heating reflux to be extracted 3 times, each 2 hours, merge extractive liquid,, filter AB8 macroporous resin on the rear filtrate, after treating that effluent is saturated, colourless with 40% alcohol flushing pillar to effluent, use 90% ethanol elution then, the fraction concentrating under reduced pressure gets gynostemma pentaphyllum herb extract 1738.3 grams.Detect with high-efficient liquid phase technique (HPLC-ELSD), the content that records chemical compound 1 and chemical compound 2 is respectively 8.9%, 12.2%.
Hydrogen spectrum data (500MHz, the C of table 1, chemical compound 1-4
5D
5N, J in Hz)
Table 2, chemical compound 1-4 carbon spectrum data (125MHz, C
5D
5N, J in Hz)
Hydrogen spectrum data (300MHz, the CDCl of table 3, chemical compound 5-8
3, J in Hz)
Hydrogen spectrum data (300MHz, the CDCl of table 4, chemical compound 9-12
3, J in Hz)
Hydrogen spectrum data (300MHz, the CDCl of table 5, chemical compound 13-15
3, J in Hz)
Test example 1: cellular level is measured the activity that chemical compound suppresses hepatic stellate cell LX2 propagation
1, the ultimate principle of mtt assay: MTT[3-(4,5)-dimethyl thiahiazo (z-y1)-3,5-di-phenytetrazoliumromide] be a kind of dyestuff of yellow color.In the living cells mitochondrion succinate dehydrogenase can metabolism reduction MTT, simultaneously under the effect of cytochrome C, (what of Formazan) ， Jia Za can be measured at the 570nm place with microplate reader to generate blue (or bluish violet) water-fast Jia Za.Under normal conditions, first Za growing amount is directly proportional with viable count, therefore can infer the number that living cells according to optical density OD value.Owing to do not contain succinate dehydrogenase in the dead cell, therefore add MTT and can not respond.
2, experiment material and instrument
LX-2 cell: buy by this laboratory;
MTT is available from Genebase;
Three liquid: 10%SDS, 5% isopropyl alcohol, 36.5%Hcl, distilled water;
Penicillin (penicillin) streptomycin (streptomycin) is available from Invitrogen 250units/ml;
DMEM culture medium: available from Invitrogen;
Hyclone (fetal bovine serum): available from GIBCO;
The sample mother solution: with chemical compound 1-15 and gynostemma pentaphyllum herb extract 16,17 usefulness 100%DMSO preparation, final concentration is 10mM, and-20 ℃ of preservations are standby;
PBS buffer: 140mM NaCl, 27mM KCl, 10mM Na
2HPO
4, 1.8mM KH
2PO
4, transfer pH to 7.4, autoclaving;
Other chemical reagent: analytical pure, available from Sigma company;
Bio-Rad ultraviolet microplate reader (Bio-Rad company).
3, experimental technique:
Collect the logarithmic (log) phase cell, after the trypsinization, culture medium is resuspended, plants in 96 orifice plates every hole 100 μ L behind the adjustment concentration of cell suspension.Cell in 96 orifice plates is placed 37 ℃, 5% CO
2Cultivated 24 hours in the incubator.Culture medium is gone in suction, will wait to sieve the sample mother solution and be made into two concentration of 5,10 μ M with complete medium, and every hole adds 100 μ L, and each sample is established 3 multiple holes, is positioned over and continues in the incubator to cultivate 72 hours, uses the mtt assay measured value.
MTT is made into 5mg/ml solution with PBS.The sample culturing that adds cell finishes the back and removes culture fluid, adds MTT (final concentration is 0.5mg/mL) the 100 μ L with the complete medium dilution, cultivates 4 hours, and then adds 100 μ L, three liquid, 37 ℃ of overnight incubation for 37 ℃.Measure the 570nm OD of place value with the Bio-Rad microplate reader.
Chemical compound is to the computing formula of LX-2 inhibition of proliferation rate:
(O
DDMSO-OD
Sample)/O
DDMSO * 100
4, experimental result
Table 6. dammarane triterpenes derivant is to hepatic stellate cell (LX-2) inhibition of proliferation activity
| Sample | Concentration | Suppression ratio (%) |
| |
10μM 5μM | 92.1 68.4 |
| Chemical compound 2 | 10μM 5μM | 67.3 33.0 |
| Chemical compound 3 | 10μM 5μM | 50.4 27.8 |
| Chemical compound 4 | 10μM 5μM | 65.3 35.3 |
| Chemical compound 5 | 10μM | 67.3 |
| Chemical compound 6 | 10μM | 65.4 |
| Chemical compound 7 | 10μM | 48.9 |
| Chemical compound 8 | 10μM | 45.6 |
| Chemical compound 9 | 10μM | 27.8 |
| Chemical compound 10 | 10μM | 25.6 |
| Chemical compound 11 | 10μM | 52.7 |
| Chemical compound 12 | 10μM | 50.9 |
| Chemical compound 13 | 10μM | 46.3 |
| Chemical compound 14 | 10μM | 45.2 |
| Chemical compound 15 | 10μM | 64.2 |
| Gynostemma pentaphyllum herb extract 16 | 10μM | 65.2 |
| Gynostemma pentaphyllum herb extract 17 | 10μM | 84.2 |
As shown in table 6, results of screening demonstration clearly dammarane's triterpenes of the present invention derivant and also have the pharmaceutical composition of this derivant to have the hepatic stellate cell of inhibition LX-2 inhibition of proliferation activity on the cellular level.We further select chemical compound 1 to make the pharmacodynamics test that following whole animal has been carried out in representative.Though following zoopery is all represented with chemical compound 1, the pharmaceutical composition that results of screening is significantly pointed out other dammarane's triterpenes derivant and contained this derivant on the above-listed cellular level also has similar effects.
Test example 2: anti-CCl
4Inductive hepatic fibrosis
1, experimental principle:
CCl
4Be converted into strong oxidizer through liver cell mitochondria, can the coup injury hepatocyte.Prolonged and repeated CCl
4Stimulation can cause the liver repair mechanism, and spider cell activates and propagation, secretes collagen deposition in a large number in extracellular matrix, forms hepatic fibrosis.CCl
4The Liver Fibrosis Model that causes is the model of present widely used evaluation anti-hepatic fibrosis medicines effect.Content reflection hepatocyte injury situation by alanine aminotransferase and aspartate transaminase in the detection mice serum; Haematoxylin-Yihong dyeing detects the pathologic condition of hepar damnification; Masson dyeing and hydroxyproline determination show liver extracellular matrix deposition situation; Immunohistochemical method detects the aSMA expression, shows star cell proliferation situation in the liver.
2, test method:
48 of male SPF level C57BL6 mices are available from Chinese Academy of Sciences's Shanghai Experimental Animal Center, body weight 20 ± 2g.Animal is divided into normal group, model group, group of solvents and 2.5mg/kg, 1mg/kg and three test group of 0.3mg/kg, 8 every group at random.Giving the medicine of 2.5mg 2.5mg/kg refer to every kilogram of mice, is chemical compound 1 here.The foundation of Liver Fibrosis Model: CCl
4Lumbar injection (1ml/kg, volume ratio 10% is dissolved in olive oil) 2 times weekly, during the injection isometric 5% ethanol, totally 4 weeks.Not modeling of normal group, only intraperitoneal injection of saline; The model group modeling is intraperitoneal injection of saline simultaneously; The group of solvents modeling is the normal saline solution of lumbar injection 1%Tween80 simultaneously; The equal modeling of test group also gives not commensurability medicine.All mice standard diets of experimental session are freely drunk water.Giving for the first time CCl
4The time begin administration, each organizes mice in last injection CCl
4Back 48h, fasting 12h is after chloral hydrate 400mg/kg anaesthetizes back execution deeply again.Collect mice serum-80 ℃ preservation.Getting the Mouse Liver middle period is dipped in 4% neutral formalin solution, makes 5 μ m paraffin sections thereafter.All the other hepatic tissues close at-80 ℃ of preservations.
The histology: the hepatic tissue specimens paraffin embedding slices is carried out haematoxylin-Yihong dyeing and Masson three collagen stainings respectively after routine dewaxing moisturizing.Activated spider cell detects through immunohistochemistry, section is through α SMA monoclonal antibody (doctor's moral company, 1:200), biotin labeled anti-mice two is anti-and after Avidin-the peroxide multienzyme complex is hatched, is the expression that substrate detects α SMA with the benzidine, dehydration, transparent and mounting are after observation by light microscope.
Serum detects: adopt Hitach i7080 automatic biochemical detector, reagent is Japan and the pure medicine of light company provides.
Hydroxyproline content is measured: build up biotech company's test kit description with reference to Nanjing and carry out, the 30mg liver adds 95 ℃ of hydrolysis 20min of 0.5mL 6mol/L NaOH, and cooling back adjust pH causes 6.8 and be settled to 2mL.Get supernatant 1mL after centrifugal and add 0.5mL chloramine-T solution, behind the room temperature 20min, add 0.5 and cross chloric acid (3.15mol/L) solution.Add 10% pair of methylamino phenenyl formaldehyde, 60 ℃ of water bath heat preservation 20min behind the 5min.The colour developing postcooling, colorimetric under spectrophotometer 550nm.Standard curve making is got 2,5,8,10,16,20 μ g respectively and is added the eppendorf pipe, adds the 0.5mL chloramine-T, and later step is with above-mentioned.According to concentration and absorbance drawing standard curve, calculate the hepatic tissue hydroxyproline content according to colourimetric number and standard curve.
Statistical analysis: The data meansigma methods scholar standard deviation is represented.Carry out statistical procedures with statistical software, relatively adopt one factor analysis of variance (one way ANOVA) between multi-group data.
3, experimental result:
(1) HE dyeing
Fig. 1: haematoxylin-Yihong (HE) colored graph of normal group, model group, group of solvents, test group (2.5mg/kg, 1mg/kg, 0.3mg/kg).
Show as Fig. 1 result: compare with normal group, model group and group of solvents have massive inflammatory cells infiltrated in lobules of liver Zhou Bianmen portal area, hepatocyte lipoid degeneration and edema necrosis, and two groups do not have significant difference; And the mouse liver that gives chemical compound 1 treatment reduces along with the increase inflammatory cell infiltration of dosage.
(2) Masson three collagen stainings
Fig. 2: the three collagen staining figure of normal group, model group, group of solvents, test group (2.5mg/kg, 1mg/kg, 0.3mg/kg) Masson.Blue expression collagen deposition position.
Show as Fig. 2 result: compare with normal group, model group and group of solvents are dyed blue collagen and are deposited on a portal area in a large number; The mouse liver blue region that gives chemical compound 1 obviously reduces, and shows that the collagen deposition in the extracellular matrix obviously reduces.
(3) α SMA immunohistochemistry
Fig. 3: group of solvents, test group (1mg/kg, 0.3mg/kg) α SMA immunohistochemistry figure.The activated spider cell of α SMA labeling section bit representation.
Show as Fig. 3 result: as seen the group of solvents section is brown xanchromatic α SMA strong positive and is present in a large number around the lobules of liver; The visible faint positive signal of 1mg/kg group section, the section of 0.3mg/kg group is weak positive, shows that group of solvents α SMA positive cell quantity is more, and it is less to give two groups of α SMA positive cells of chemical compound 1.
(4) measure mice serum alanine aminotransferase and aspartate transaminase content
Fig. 4: normal group, model group, group of solvents, test group (2.5mg/kg, 1mg/kg, 0.3mg/kg) serum aspartate transaminase (A) and alanine aminotransferase (B) content sketch map, * represents P＜0.05.
Measurement result shows: chemical compound 1 can significantly reduce CCl
4The content of liver fibrosis due mice serum alanine aminotransferase and aspartate transaminase shows that chemical compound 1 has the hepatocellular effect of protection.
(5) measure the hepatic tissue hydroxyproline content
Fig. 5: normal group, model group, group of solvents, test group (2.5mg/kg, 1mg/kg, 0.3mg/kg) hepatic tissue hydroxyproline content sketch map, * represents P＜0.05.
Test result shows: chemical compound 1 can significantly reduce CCl
4The content of hydroxyproline in the liver fibrosis due murine liver tissue shows that chemical compound 1 has the effect that suppresses collagen deposition in the extracellular matrix.
By above result as can be known, chemical compound 1 can reduce the content of model mice serum alanine transaminase and aspartate transaminase, suppresses hepatic stellate cell propagation, reduces the extracellular matrix collagen deposition, to CCl
4The hepatic fibrosis mouse model that causes has anti-fibrosis effect.
Above embodiment and test example have only provided part of compounds, its preparation method and the pharmacodynamic experiment result of dammarane's triterpenes derivant of the present invention, but can make various modifications and variation to this to one skilled in the art, and not deviating from the spirit and scope of the present invention, appending claims covers all such modifications in the scope of the invention.
Claims (6)
1, the application of dammarane's triterpenes derivant in preparation prevention or the hardened medicine of treatment hepatic fibrosis regulating liver-QI of representing by following general formula (I),
Wherein, R
1Be selected from H, OH, carbonyl (O=), C
1-C
10Acyloxy or arabinose, galactose, glucose, mannose, rhamnose, ribose, xylose, or the oligonucleotide chain formed by glycosidic bond of the arabinose of esterification, galactose, glucose, mannose, rhamnose, ribose, xylose and at least two sugar or sugar ester; R
2Be selected from C
1-C
5Alkyl or C
1-C
5Thiazolinyl.
2, according to the application of dammarane's triterpenes derivant of claim 1, it is characterized in that: the described dammarane's triterpenes of general formula (I) derivant is any in following 15 chemical compounds:
3, a kind of pharmaceutical composition of anti-hepatic fibrosis, it comprises dammarane's triterpenes derivant of claim 1 general formula (I) expression.
4, a kind of pharmaceutical composition of anti-hepatic fibrosis, it comprises the gynostemma pentaphyllum herb extract, described gynostemma pentaphyllum herb extract contains dammarane's triterpenes derivant of claim 1 general formula (I) expression.
5, according to the pharmaceutical composition of claim 4, wherein said gynostemma pentaphyllum herb preparation method of extract is as follows: gynostemma pentaphyllum herb is added water boil extract, AB8 macroporous resin on the extracting liquid filtering rear filtrate, after treating that effluent is saturated, water flushing pillar is colourless to effluent, use 90% ethanol elution then, the fraction concentrating under reduced pressure promptly gets the gynostemma pentaphyllum herb extract.
6, the application of each described pharmaceutical composition in preparation prevention or the hardened medicine of treatment hepatic fibrosis regulating liver-QI among the claim 3-5.
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| CN2008100346682A CN101530419B (en) | 2008-03-14 | 2008-03-14 | Application of dammarane triterpenes derivative and drug composition containing dammarane triterpenes derivative |
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| CN2008100346682A CN101530419B (en) | 2008-03-14 | 2008-03-14 | Application of dammarane triterpenes derivative and drug composition containing dammarane triterpenes derivative |
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| CN101530419A true CN101530419A (en) | 2009-09-16 |
| CN101530419B CN101530419B (en) | 2011-02-16 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103012537A (en) * | 2012-12-20 | 2013-04-03 | 上海交通大学 | Dammarane type triterpene compound and preparation method and application thereof |
| CN105267196A (en) * | 2015-11-09 | 2016-01-27 | 南京大学 | Composition and application of composition to anti-hepatic fibrosis drugs |
| CN105326842A (en) * | 2015-10-14 | 2016-02-17 | 南京大学 | Composition and application of composition to preparation of medicines for treating or preventing renal fibrosis |
| CN115368425A (en) * | 2021-08-06 | 2022-11-22 | 遵义医科大学 | Triterpenoid compound, preparation method and anti-inflammatory application thereof |
-
2008
- 2008-03-14 CN CN2008100346682A patent/CN101530419B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103012537A (en) * | 2012-12-20 | 2013-04-03 | 上海交通大学 | Dammarane type triterpene compound and preparation method and application thereof |
| CN103012537B (en) * | 2012-12-20 | 2014-10-29 | 上海交通大学 | Dammarane type triterpene compound and preparation method and application thereof |
| CN105326842A (en) * | 2015-10-14 | 2016-02-17 | 南京大学 | Composition and application of composition to preparation of medicines for treating or preventing renal fibrosis |
| CN105267196A (en) * | 2015-11-09 | 2016-01-27 | 南京大学 | Composition and application of composition to anti-hepatic fibrosis drugs |
| CN115368425A (en) * | 2021-08-06 | 2022-11-22 | 遵义医科大学 | Triterpenoid compound, preparation method and anti-inflammatory application thereof |
| CN115368425B (en) * | 2021-08-06 | 2023-07-18 | 遵义医科大学 | Triterpene compound, preparation method and anti-inflammatory application thereof |
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| Publication number | Publication date |
|---|---|
| CN101530419B (en) | 2011-02-16 |
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